Many antibodies, ligand receptors, regulatory enzymes (e.g., kinases, glycosyltransferases, and lipid transferases), and other biomolecules are attractive therapeutic targets. Antigens, ligands, peptides, substrates, and small molecules that bind to, activate, and/or inhibit these biomolecules are considered to be potential drug candidates. High-throughput screening of combinatorial libraries of potential drug candidates is pivotal to the identification of large numbers of lead compounds for drug development.
Methods of screening for reagents that bind to, and modulate, biomolecules can be divided into two broad categories: homogeneous methods and separation-based methods. In homogeneous methods, the formation of a reaction product is monitored without its separation from any unreacted reagents. In separation-based methods, the reaction product is separated from any unreacted reagents by chromatography (e.g., capillary chromatography) or electrophoresis (e.g., capillary electrophoresis), prior to detection. An “ideal” method for high-throughput screening should require only nanoliter volumes of the biomolecule and the candidate drug.
Microreactors, which facilitate chemical processes in nanoliter and sub-nanoliter volumes, are highly attractive for high-throughput screening (Steger et al., The dispensing well plate: a novel device for nanoliter liquid handling in ultra high-throughput screening. Journal of the Association for Laboratory Automation, 9(5):291-99, 2004) and a variety of other applications, including multiplexed bioanalyses (Moser et al., Microsphere sedimentation arrays for multiplexed bioanalytics. Analytica Chimica Acta, 558:102-09, 2006), studies of single molecules (Lee et al., Single-molecule spectroscopy for molecular identification in capillary electrophoresis. J. Chromatogr., A., 1053:173-79, 2004), and analyses of the chemical contents of single cells (Hellmich et al., Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology. Electrophoresis, 26:3689-696, 2005). The primary requirements for microreactors include: (i) easy and reproducible mixing, (ii) negligible evaporation, and (iii) interfacing with sensitive and informative analytical tools. These requirements can be met, for example, by confining the nanoliter-volume reaction mixture in a microfabricated well (Chang et al., A microfabricated device for the characterization of biological species. Journal of Vacuum Science and Technology B, 20(5): 2058-064, 2002), oil drop (Hiroyauki, Noji, Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology, Nature Biotech., 23:361-65, 2005), or capillary format (Shen et al., Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell, Electrophoresis, 22(17):3677-682).
The known formats for microreactors have their specific advantages and limitations. For example, the well and oil-drop formats require precise microprinting instrumentation for accurate mixing of reactants. Additionally, the well format may require complicated technological solutions for closing the wells to prevent evaporation. Both of these formats are not easily interfaced with separation techniques and are limited to in situ optical detection. Furthermore, these two formats require either the use of relatively noisy fluorophore-quencher systems for monitoring non-covalent binding or the use of rare fluorogenic substrates for studying enzymatic reactions.
Capillaries perfectly suit the evaporation and analysis requirements of microreactors. Indeed, due to the extremely small liquid-air interface at the capillary orifice, evaporation can be neglected for as long as days. In addition, capillary microreactors are naturally interfaced with highly-efficient analytical techniques, such as capillary electrophoresis and chromatography. In turn, capillary separation can be readily interfaced with different types of detection, including optical, electrochemical, and mass spectrometric detection, thereby providing ultimate analytical capabilities. However, to date, the challenge that has prevented the widespread practical application of capillary microreactors has been the requirement for easy and reproducible mixing of reactants.
Two methods have been proposed for mixing reactants inside capillaries: electrokinetic mixing and mixing by longitudinal diffusion. However, as discussed below, neither of these methods provides a generic way of mixing reactants in a capillary, which is required for practical application of capillary microreactors.
Electrokinetic mixing is based on different velocities of reactants in an electric field applied to the ends of a capillary (Hiroyauki, N., Nature Biotech., 23:361-65, 2005). This mode of mixing requires knowledge of the electrophoretic mobilities of the reactants; this cannot be calculated, and should be experimentally determined. Electrokinetic mixing becomes impractical when the reactants are dissolved in different buffers, or when three or more reactants are to be mixed.
Mixing by longitudinal diffusion is based on diffusion through transverse interfaces between separately-injected plugs of reactants (Okhonin et al., Transverse diffusion of laminar flow profiles to produce capillary nanoreactors. Anal. Chem., 77:5925-929, 2005). The characteristic length of an injected plug is 1 mm; several plugs have a cumulative length of several millimetres. Longitudinal diffusion through several millimetres can take as long as several hours. Thus, longitudinal diffusion is impractical for a typical geometry of plugs. Furthermore, it is inapplicable to the mixing of three or more reagents inside a capillary (see, e.g., Taga and Honda, J. Chromatogr., A., 742:243-50, 1996).
Due to the lack of a generic approach to mixing multiple reagents inside a capillary, capillary separation has, until now, required pre-mixing components in a vial outside of the capillary. This limits the minimum volume of reagents consumed per analysis to approximately 1 μL—three orders of magnitude greater than the requirement. This results in wastage of the reagents. Additionally, if mixing by longitudinal diffusion is performed for studies of reactions, the reactions can proceed to a significant extent because longitudinal diffusion takes a very long time; this will prevent separate modeling of mixing and reaction kinetics. Simultaneous modeling of mixing and reaction kinetics is much more demanding, as it requires the knowledge of rate constants, which are often not available.
Accordingly, in view of the foregoing, there exists in the art a need for a separation-based method of drug screening that requires only nanoliter or subnanoliter volumes of reagents and permits two or more reagents to be mixed inside a capillary. There also exists in the art a need for a universal numerical model which accurately simulates mixing of two or more reagents inside a capillary.